Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-12-21
2004-07-13
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007720, C436S510000, C436S517000, C436S518000, C436S808000, C436S065000
Reexamination Certificate
active
06762033
ABSTRACT:
The invention relates to a method for determining the fertility of mammals, in particular of humans.
Due to the increase in the knowledge and abilities concerning the in vitro-fertilization technique, the demand of reliable and also low-cost fertility determining methods has highly increased. What is involved is not merely the basic diagnosis of fertility disturbances as such, but increasingly also the detection of the degrees of fertility and the determination of the fertilization and/or nidation of the fertilized oocytes.
Afamin is a 87 kDa protein belonging to the albumin group and having many things in common, structurally and in terms of biochemistry, with the proteins of this group, such as, e.g., with human serum albumin, human &agr;-fetoprotein or human vitamin D binding protein. Afamin has already been cloned and sequenced and thus is also available in recombinant form (WO 95/27059).
Moreover, biochemical and physiological tests have shown that this protein has vitamin E-binding properties. It could be demonstrated that afamin occurs not only in blood, but also in other body and organ fluids, such as, e.g., cerebrospinal, follicular and seminal fluid.
The present invention has as its object to provide a completely novel fertility determination method.
According to the invention, this object is achieved by a method for determining the fertility of mammals, in particular humans, which is characterized in that
a body or organ fluid is taken from a mammal,
the afamin content is determined in this body or organ fluid, and
the determined afamin content is compared with a reference value so as to determine the fertility.
The present invention is based on the surprising finding that the afamin concentration in the various body or organ fluids, where afamin is present, directly correlates with the fertility properties. This correlation is not merely restricted to the presence of fertility disturbances, but is also possible for diagnosing fertility fluctuations or pregnancies. According to the invention, it has even been shown that apart from the detection of the presence or absence of oocytes via the methodology according to the invention for determining the afamin content, even an evaluation of the degree of maturity of oocytes is possible.
The present invention may be used in human medicine, in particular in the monitoring of in vitro fertilizations or in fertilization diagnoses and expert opinions. Yet it also has enormous possibilities within the scope of modern animal breeding, since it is easy to standardize and does not require complicated laboratory equipment for carrying out the tests.
Afamin is present in many different body or organ liquids, and it has been shown according to the invention that the afamin content in all these fluids correlates with the fertility properties. According to preferred embodiments of the present invention, however, the afamin content is primarily determined in body or organ fluids which are characterized by a high physiological afamin content, such as, e.g., serum, follicular or seminal fluid. Yet it is, of course, also possible to carry out the method according to the invention with other body or organ fluids, such as cerebrospinal fluid, since the concentration of afamin in these other fluids also lies in a range which, as a rule, does not cause any problems in terms of the possible afamin detection limit.
In practice, the method according to the invention may be carried out in any manner desired, primarily as regards the manner of taking the body or organ fluid or as regards the determination of the afamin content. The afamin content may, e.g. be determined immunologically, electrophoretically or chromatographically. According to the invention, immunological determination methods often are preferred for afamin, since not only a series of different monoclonal antibodies are available against different epitopes of afamin, but because it is particularly immunological tests, such as in the form of standard ELISA tests, which are easy to design such that they can also be carried out and evaluated without any complex laboratory instruments (e.g. in combination with calorimetric detection methods). In this manner it is possible to provide the determination method according to the invention also in a form which is easy to carry out for common people.
As the reference value, usually an afamin normal value for the respective body or organ fluid is used. In the present method, this may, e.g., be obtained in the form of comparative values, comparative curves or comparative tables or—as is generally preferred—by a simultaneous determination of a reference sample (having a defined afamine content) together with the sample of the body or organ fluid taken. In the latter instance, the systematic error possibly resulting from using different determination methods of different determination conditions, which probably can never be completely eliminated, is avoided right from the beginning. This may be particularly important in determinations where merely gradual differences in afamin contents (e.g. when determining the degree of maturity of oocytes) are at issue.
Preferably, the sample measured according to the invention is not only compared with one reference value, but it is compared with two or more reference values. Thus it is, e.g., possible to provide a different reference value or a reference sample, such as, e.g., a “pathological” reference or a “pregnancy” reference, etc., besides a “normal value”.
According to the invention, however, it is preferred to provide a reference value for the afamin content in the corresponding body or organ fluid which corresponds to the afamin content of a normal patient (or, in animal breeding, to the sample of the normal animal).
When carrying out the method according to the invention, this reference value preferably is obtained in that the afamin content of a reference sample is determined in parallel with the fertility determination of the sample.
According to the invention, the determination of the afamin content preferably is effected by using immunological methods, in particular by using a monoclonal antibody, since by this standardizing can be achieved very efficiently and also for the most varying lots of a determination kit, the data found will be compatible among themselves.
According to the invention it has been shown that particularly in the seminal and follicular fluid, beside the afamin content, also the vitamin E content directly correlates with the fertility property and thus likewise may be employed for the method according to the invention. Thus, the method according to the invention preferably is further combined with a determination of the vitamin E content in the respective body or organ fluid, which vitamin E content then optionally also will be compared with a reference value.
Numerous methods of determining vitamin E in body or organ fluids have been described. As in the determination of the afamin content within the scope of the present invention, the specific manner in which the vitamin E content is determined likewise is not critical for the present invention, yet HPLC or other biochemical methods are the preferred methods of determining vitamin E concentrations.
In a further aspect, the present invention relates to the use of afamin for determining the fertility properties of mammals.
A further aspect of the present invention relates to a kit for determining the fertility of mammals, which comprises
a sample of a body or organ fluid from a mammal, or a vessel for receiving the body or organ fluid,
a reagent for detecting the afamin content in the sample, and
afamin reference means.
As mentioned above, the choice of the reagent for detecting the afamin content will, of course, depend on the respective detection methodology used. For instance, the reagent for detecting the afamin content preferably comprises an antibody to afamin, in particular a monoclonal afamin antibody. Preferably, this afamin antibody also comprises further detection means, such as fluorescent, radioactive or chromog
Dieplinger Hans
Illmensee Karl Oskar
Davis Deborah A
Fulbright & Jaworski L.L.P.
Le Long V.
Vitateq Biotechnology GmbH
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